CN110343175A - Inhibit the scFv sequence and its application of HIV-1 virus infection - Google Patents

Inhibit the scFv sequence and its application of HIV-1 virus infection Download PDF

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CN110343175A
CN110343175A CN201910558818.8A CN201910558818A CN110343175A CN 110343175 A CN110343175 A CN 110343175A CN 201910558818 A CN201910558818 A CN 201910558818A CN 110343175 A CN110343175 A CN 110343175A
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CN110343175B (en
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张辉
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Shenzhen Renewal City Biomedical Technology Co Ltd
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Shenzhen Renewal City Biomedical Technology Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Abstract

The invention discloses the scFv sequences and its application that inhibit HIV-1 virus infection.Variable region heavy (V based on HIV-1 neutralizing antibody VRC01H) and light chain (VL), its sequence is connected to form scFv using own method, 9 scFv molecules that there is higher affinity with the gp120 albumen of HIV-1 are obtained.The scFv sequence of certain embodiments of the present invention has good affinity to HIV-1 virus, can effectively inhibit infection of the virus to target cell TZM-bl cell.It using the scFv sequence of certain embodiments of the present invention, can construct to obtain the CAR-T cell that can inhibit HIV-1 virus infection, which can forcefully mediate the cracking of target cell, discharge LDH.The CAR-T cell of certain embodiments of the present invention can preferably avoid escape caused by HIV-1 virus mutation, have the HIV-1 HIV suppression effect of more wide spectrum by loading a variety of CAR molecules with different scFv sequences.

Description

Inhibit the scFv sequence and its application of HIV-1 virus infection
Technical field
The present invention relates to cellular immunotherapy technical fields, and in particular to inhibit HIV-1 virus infection scFv sequence and It is applied.
Background technique
After human immunodeficiency virus type 1 (Human ImmunodificiencyVirusType-1, HIV-1) infection, connection Virus can be effectively inhibited again by closing antiretroviral therapy (combined antiretroviral therapy, cART) System.However, in infected cell and forming a stable latent infection repository due to viral integrase, it is infected person once Stop cART treatment, viremia virusemia is broken out again in a short time, and which constitute the major obstacles for curing HIV-1 infection.
Current research hotspot is the latent infection reversing drug (latency-reversing by specificity Agents, LRAs) activating dormant infection virus, and then drug therapy or induction body immune system kill infected cell. This Intervention Strategy is referred to as " shockandkill ".However, HIV-1 can rapidly mutate to escape Immune discrimination. Researches show that in the infected by cART treatment, even if its latent infection of successful activation, intracorporal CD8+T lymph is thin Born of the same parents are due to lacking to the effective immune response of HIV-1, so cannot fully erased infected cell.Therefore, exist In " shockandkill " strategy, in order to preferably remove latent infection repository, needing to rebuild in infected person's body has by force The immunosurveillance function of power.
In recent years, since with high-affinity, TCR (T cell receptor) is non-dependent and MHC (major Histocompatibility complex) it is unrestricted the features such as, Chimeric antigen receptor (chimeric antigen Receptor, CAR) immune cell therapy become the completely new approach of killing tumor cell.CAR be by antibody target region with T cell activation intracellular signal area is merged, to assign cell-specific antigens recognition capability.By immune in autologous patient In cell expression identification tumour native antigen CAR molecule and to carry out adoptive immunity feedback, can specificity target killing The tumour cell of patient's body.CAR-T cell therapy demonstrates validity in the clinical treatment of leukaemia and lymthoma, and And obtain encouraging success.The strategy can also be applied to antiviral therapy, including HIV-1, HBV (hepatitis B ) and the treatment of the virus infections such as HCV (hepatitis C virus) virus.But the CAR molecule of conventional construction can't be complete The immunization therapy of the full up various diseases of foot.
In early-stage study, inventor is by the of the scFv sequence in the source HIV-1 wide spectrum neutralizing antibody VRC01 and brand-new design Intracellular section of sequence of three generations's CAR molecule links together and transduction of CD 8+T cell, the VC-CAR-T for producing HIV-1 specificity are thin Born of the same parents.Compared to reported CD4-CAR-T cell, the cell-mediated specific killing effect of VC-CAR-T is more obvious.And for the first time Latent infection repository (the Liuet being activated in cART treatment HIV-1 the infected's sample can effectively be removed by demonstrating the therapy al,Journal of Virology,90:9712-9724.2016)。
But HIV-1 mutation rate with higher, to generate escape clone.It is necessary to develop more to HIV-1 disease Poison infects inhibited molecule.
Summary of the invention
It is an object of the invention to overcome at least one deficiency of the prior art, provides and inhibit HIV-1 virus infection ScFv sequence and its application.
The technical solution used in the present invention is:
The first aspect of the invention provides:
Inhibit the scFv albumen of HIV-1 virus infection, the sequence of the scFv albumen such as SEQ ID NO.:1~SEQ ID Shown in NO.:9.
The second aspect of the invention provides:
A kind of CAR molecule inhibiting HIV-1 virus infection, the CAR molecule includes extracellular antigen binding domain and knot intracellular Structure domain, the extracellular antigen binding domain are scFv albumen, the sequence of the scFv albumen such as SEQ ID NO.:1~SEQ ID Shown in NO.:9.
In the example of some CAR molecules, the sequence of the intracellular domain is as shown in SEQ ID NO.:10.
The third aspect of the invention provides:
A kind of CAR-T cell inhibiting HIV-1 virus infection, the CAR-T cell transduction have at least one CAR molecule, The CAR molecule includes extracellular antigen binding domain and intracellular domain, and the extracellular antigen binding domain has SEQ ID NO.:1 Albumen shown in~SEQ ID NO.:9 any sequence.
In the example of some CAR-T cells, the extracellular antigen binding domain has SEQ ID NO.:1, SEQ ID Sequence shown in NO.2 and SEQ ID any one of NO.5.
In the example of some CAR-T cells, the sequence such as SEQ of the intracellular domain of the CAR molecule of the CAR-T cell Shown in ID NO.:10.
In the example of some CAR-T cells, T cell CD8+T cell.
The fourth aspect of the invention provides:
Application of the CAR-T cell in the preparation that HIV-1 infection cell is removed in preparation, the CAR-T cell such as present invention Third in terms of described in.
The fifth aspect of the invention provides:
Application of the scFv albumen in the CAR-T cell that building inhibits HIV-1 virus infection, the sequence of the scFv albumen As shown in one of SEQ ID NO.:1~SEQ ID NO.:9.
The sixth aspect of the invention provides:
ScFv albumen is preparing the application in HIV-1 virus targeting preparation, the sequence of the scFv albumen such as SEQ ID Shown in one of NO.:1~SEQ ID NO.:9.
The beneficial effects of the present invention are:
The scFv sequence of certain embodiments of the present invention has good affinity to HIV-1 virus, can effectively inhibit virus Infection to target cell TZM-bl cell.
Using the scFv sequence of certain embodiments of the present invention, can construct to obtain the CAR-T that can inhibit HIV-1 virus infection Cell, the CAR-T cell can forcefully mediate the cracking of target cell, discharge LDH.
The CAR-T cell of certain embodiments of the present invention can be with by loading a variety of CAR molecules with different scFv sequences Escape caused by HIV-1 virus mutation is preferably avoided, there is the HIV-1 HIV suppression effect of more wide spectrum.
Detailed description of the invention
Fig. 1 is that QY-PhD-001 to the QY-PhD-009 molecule filtered out carries out HIV-1 virus neutralization experiment result；
Fig. 2 is the QY-PhD-CAR schematic arrangement of building；
Fig. 3 is the VC-CAR schematic arrangement of building；
Fig. 4 is the CD8+T cell and table of QY-PhD-001-CAR, QY-PhD-002-CAR and QY-PhD-005-CAR transduction Up to after the target cell system mixed culture of HIV-1 envelope protein gp120, by detecting the release of LDH, detect to purpose target cell Killing activity, VC-CAR-T cell are positive control, and HER2-CAR-T cell is negative control；
Fig. 5 is QY-PhD-001-CAR, QY-PhD-002-CAR and QY-PhD-005-CAR cell and does not express purpose egg After white control target cell system mixed culture, by detecting the release of LDH, the killing activity to purpose target cell, VC- are detected CAR-T cell is positive control, and HER2-CAR-T cell is negative control.
Specific embodiment
The first aspect of the invention provides:
Inhibit the scFv albumen of HIV-1 virus infection, the sequence of the scFv albumen such as SEQ ID NO.:1~SEQ ID Shown in NO.:9.
The second aspect of the invention provides:
A kind of CAR molecule inhibiting HIV-1 virus infection, the CAR molecule includes extracellular antigen binding domain and knot intracellular Structure domain, the extracellular antigen binding domain are scFv albumen, the sequence of the scFv albumen such as SEQ ID NO.:1~SEQ ID Shown in NO.:9.
In the example of some CAR molecules, the sequence of the intracellular domain is as shown in SEQ ID NO.:10.
The third aspect of the invention provides:
A kind of CAR-T cell inhibiting HIV-1 virus infection, the CAR-T cell transduction have at least one CAR molecule, The CAR molecule includes extracellular antigen binding domain and intracellular domain, and the extracellular antigen binding domain has SEQ ID NO.:1 Albumen shown in~SEQ ID NO.:9 any sequence.
In the example of some CAR-T cells, the CAR-T cell can load a variety of CAR molecules, to be embodied as wide spectrum Specific killing action.
In the example of some CAR-T cells, the extracellular antigen binding domain has SEQ ID NO.:1, SEQ ID Sequence shown in NO.2 and SEQ ID any one of NO.5.
In the example of some CAR-T cells, the sequence such as SEQ of the intracellular domain of the CAR molecule of the CAR-T cell Shown in ID NO.:10.
In the example of some CAR-T cells, T cell is CD8+T cell.
The fourth aspect of the invention provides:
Application of the CAR-T cell in the preparation that HIV-1 infection cell is removed in preparation, the CAR-T cell such as present invention Third in terms of described in.
The fifth aspect of the invention provides:
Application of the scFv albumen in the CAR-T cell that building inhibits HIV-1 virus infection, the sequence of the scFv albumen As shown in one of SEQ ID NO.:1~SEQ ID NO.:9.
The sixth aspect of the invention provides:
ScFv albumen is preparing the application in HIV-1 virus targeting preparation, the sequence of the scFv albumen such as SEQ ID Shown in one of NO.:1~SEQ ID NO.:9.
Specifically, can be by scFv albumen and other molecule coupling labeleds, using scFv albumen to the gp120 albumen of HIV-1 High-affinity realizes detection, drug molecule Targeting delivery etc. to internal HIV-1 virus.
Below with reference to experiment, technical solution of the present invention is further illustrated.
The acquisition of scFv
The present invention is based on the Variable region heavy (V of HIV-1 neutralizing antibody VRC01H) and light chain (VL), it will using own method Its sequence is connected to form scFv, be obtained with the gp120 albumen of HIV-1 have higher affinity 9 scFv molecules, and by its It is named as QY-PhD-001 to QY-PhD-009 (its amino acid sequence respectively corresponds NO.:1~9 SEQ ID).
SEQ ID NO.:1 is QY-PhD-001 molecule
EIVLTQSPGTLSLSPGETAIISCRSLWYQQRPGQAPRLVIYLQYPAGPLLGIPDRFSGSRWGPDYNLTI SNLESGDFGVYYCRLEGSPPVNFGQGTKVQVDIKRQVQLVQSGGQMKKPGESMRISCRASRSPTRNSHRLARLLHLT PRLHQLTEPPPPEEPPPPEEPPPPDRLMSTWTLVPWPNTGTARQTPKSPDSRLLMVRLSGPHRLPLNLSGMPLAING GIMTAWGPGRASVDTSGSPWGVTRDKRWLFPLETKTGCLETVSTQFPWPAGPHRKEQLKELRIIIFSRKSPKKRLQS LASWLFPVNCYPLTIPHNIRAGSIKGKAWGAWGRGTPVIVSS
SEQ ID NO.:2 is QY-PhD-002 molecule
EIVLTQSPGTLSLSPGETAIISCSPVVMQWYQQRPGQAPRLVIYYGFIPSGLASIGIPDRFSGSRWGPD YNLTISNLESGDFGVYYCGTPGRYREPVFGQGTKVQVDIKRQVQLVQSGGQMKKPGESMRISCRASRSPTRNSHRLA RLLHLTPRLHQLHLRAAAARRAAAARRAAAARSLNVDLDLGPLAKSSSYCAIINTKISRLQVADGEIVVWPHRLPLN LSGMPMCIQRREMTSLGAWPGLCYHADLLKGDTQEMMAVSPGDKDRVPGDCVKQFPWPAGPHRKEQLKELRIIASVD TTQTCRGETHKRWLFPLETKTGCLETVSNNFPWPSLGRKRKEQLEEWGRGTPVIVSS
SEQ ID NO.:3 is QY-PhD-003 molecule
EIVLTQSPGTLSLSPGETAIISCWLCWYQQRPGQAPRLVIYYHLALRPFPRGGIPDRFSGSRWGPDYNL TISNLESGDFGVYYCGIVSPLEARQFGQGTKVQVDIKRQVQLVQSGGQMKKPGESMRISCRASEILIDSPGFFIPPD CTSCTEPPPPEEPPPPEEPPPPDRLMSTWTLVPWPNRRLIAQTPKSPDSRLLMVRLSGPTDCRGTCLGCRCVSSDEN RRAWGPGRASVDTTQTCRGETHKRWLFPLETKTGCLETVSNNFHGRLGRIERNNRNCEFFHVENLQKEAPSLASWSL VPEWGRGTPVIVSS
SEQ ID NO.:4 is QY-PhD-004 molecule
EIVLTQSPGTLSLSPGETAIISCWRSWYQQRPGQAPRLVIYLFQIKPPGIKLGGIPDRFSGSRWGPDYN LTISNLESGDFGVYYCTIRLLKKPDKFGQGTKVQVDIKRQVQLVQSGGQMKKPGESMRISCRASKFSSTRQASSSDP QTAPAAPESRRRQKSRRRQSLNVDLDLGPLAKRGLNYAIINTKISRLQVADGEIVVWPPPTAAEPVWDAKKGLLMKI DDEPGGLAGPLLIPAPRNVLTGTRDDGCFPWRQRQGAWRLCQHNSMAGWAAKGTTKGIANNNFFTLKISKKKAPKLG VIMVIAVSCVKLLSAHNSTQHTSRKHKVSLGCLMSELTHWGRGTPVIVSS
SEQ ID NO.:5 is QY-PhD-005 molecule
EIVLTQSPGTLSLSPGETAIISCFCVCEWYQQRPGQAPRLVIYHGHSFRASRLVGGIPDRFSGSRWGPD YNLTISNLESGDFGVYYCTNPSIKLETPFGQGTKVQVDIKRQVQLVQSGGQMKKPGESMRISCRASPPKRSPTRNSH RLARLLHLTPRLHQLHLRAAAARRAAAARRAAAARSLNVDLDLGPLAKPLGLNAIINTKISRLQVADGEIVVWPPPT AAEPVWDAPWPGGEAIDDEPGGLAGPLLISLLCVRLKATRDDGCFPWRQRQGAWRLCQHNFHGRLGRIERNNRNCEF FHVENLQKKGSKAWRNHGHSCFLCEIVIRSQFHTTYEPEASVKPGVWGRGTPVIVSS
SEQ ID NO.:6 is QY-PhD-006 molecule
EIVLTQSPGTLSLSPGETAIISCSGHRWYQQRPGQAPRLVIYYHLALRPFPRGGIPDRFSGSRWGPDYN LTISNLESGDFGVYYCRIQALQVGIVFGQGTKVQVDIKRQVQLVQSGGQMKKPGESMRISCRASSPLEARQEILIDS PGFFIPPDCTSCTEPPPPEEPPPPEEPPPPDRLMSTWTLVPWPNRRLIAQTPKSPDSRLLMVRLSGPTDCRTCLGCR CVSSDENRRAWGPGRASVDTTQTCRGETHKRWLFPLETKTGCLETVSNNFHGRLGRIERNNRNCEFFHVENLQKRGS KAWRNHGHSCFLCEIVIRSQFHTTYEPEASWGRGTPVIVSS
SEQ ID NO.:7 is QY-PhD-007 molecule
EIVLTQSPGTLSLSPGETAIISCSGHRLFFLWYQQRPGQAPRLVIYHSVVSLDALLIGGIPDRFSGSRW GPDYNLTISNLESGDFGVYYCGGTRRVKRSPFGQGTKVQVDIKRQVQLVQSGGQMKKPGESMRISCRASTRNSHRLA RLLHLTPRLHQLHLRAAAARRAAAARRAAAARSLNVDLDLGPLAKFPVSAIINTKISRLQVADGEIVVWPPPTAAEP VWDAYNAKVEGIDDEPGGLAGPLLIPSRVSSNISLTRDDGCFPWRQRQGAWRLCQHNFHGRLGRIERNNRNCEFFHV ENLQKKGSKAWRNHGHSCFLCEIVIRSQFHTTYEPEASVKPGVPWGRGTPVIVSS
SEQ ID NO.:8 is QY-PhD-008 molecule
EIVLTQSPGTLSLSPGETAIISCETGGWYQQRPGQAPRLVIYESWSLFQIKPPGGIPDRFSGSRWGPDY NLTISNLESGDFGVYYCTYRVTIRLLKFGQGTKVQVDIKRQVQLVQSGGQMKKPGESMRISCRASKPDKKFSSTRQA SSSDPQTAPAAPESRRRQKSRRRQSLNVDLDLGPLAKRGLNYAIINTKISRLQVADGEIVVWPPPTAAEPVWDAKKG LLMKIDDEPGGLAGPLLIPAPRNVLTGTRDDGCFPWRQRQGAWRLCQHNSMAGWAAKGTTKGIANNNFFTLKISKKK APKLGVIMVIAVSCVKLLSAHNSTQHTSRKHKVSLGCLMSELTHWGRGTPVIVSS
SEQ ID NO.:9 is QY-PhD-009 molecule
EIVLTQSPGTLSLSPGETAIISCDWWSWYQQRPGQAPRLVIYKAARYKTRNPRGGIPDRFSGSRWGPD YNLTISNLESGDFGVYYCQGDDPAPQEAFGQGTKVQVDIKRQVQLVQSGGQMKKPGESMRISCRASRQEILIDSPG FFIPPDCTSCTEPPPPEEPPPPVACRPGPWSPGQTWAQLCNNKHQNLPTPGCWDCSLAPTDCRTCLGCQKRITNED RRAWGPGRASVDTSSSERTDLGYKRWLFPLETKTGCLETVSTQFHGRLGRIERNNRNCEFFHVENLQKKGSKAWRN HGHSCFLCEIVIRSQFHTTYEPEASVKPGVPNEANSWGRGTPVIVSS。
The Gene therapy of scFv molecule is tested:
By obtain 9 scFv sequences, it is connected to the eukaryocyte table with antibody constant region (Fc) and secreting signal peptide Up on carrier, after the correctness by sequencing confirmation sequence, HEK293T cell is transfected.The supernatant of Transfected cells is collected, and is led to After crossing ELISA titration, antibody concentration is determined.
Utilize HIV-1NL4-3Wild type infection TZM-bl cell is carried out in antibody and is tested, wide spectrum neutralizing antibody VRC01 further determines that the antibody sequence that screening obtains by measuring the readings of infected cell luciferase as positive control Whether column have antiviral functions.In this 9 sequences, inventors have found that wherein three sequences inhibit the function of virus infection It protrudes the most, respectively QY-PhD-001, QY-PhD-002, QY-PhD-005 (see Fig. 1).
The building of QY-PhD-CAR-T cell
Will determining three QY-PhD with more preferable antiviral neutralization of experiment scFv sequence and CAR molecule across Spanning domain is connected with intracellular domain, and the end CAR molecular sequences C ' is including the CD28 based on three generations's CAR structure (nucleotides 460-660, GenBank NM_006139.3), CD137 (nucleotides 640-765, GenBank ) and CD3 ζ (nucleotides 160-492, Genbank NM_198053.2) intracellular domain series connection group NM_001561.5 At scFv is connected with intracellular signal molecule by the transmembrane domain of CD28 molecule.I.e. SEQ ID NO.:1, SEQ ID NO.:2, SEQ ID NO.:5 connect with SEQ ID NO.:10 respectively, to provide one group of completely new CAR molecule, name respectively For QY-PhD-001-CAR, QY-PhD-002-CAR, QY-PhD-005-CAR, the QY-PhD-CAR of synthesis by NheI and EcoRI double digestion, be inserted into pCPPT-IRES-mStrewbeery slow virus carrier in, digestion, connection specific steps with reference to this The conventional method in field；PCPPT-QY-PhD-CAR-IRES- after obtaining QY-PhD-CAR molecule (Fig. 2) recombination MStrewbeery slow virus carrier.
SEQ ID NO.:10 is the intracellular domain signaling molecule of CAR molecule
GGGGSGGGGSGGGGSFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA CDIYIWAPLAGTCGVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRFSVV KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDV LDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALP PRDYKDDDDK。
The HEK293T cell of one growth conditions is averagely laid in the 10cm culture dish handled with poly-D-lysine (thin Born of the same parents' density is about 6.5 × 104/cm2), it is desirable that cell is in individually to be uniformly distributed.After culture about 24 hours, cell confluency degree should be approached 80%, each ware changes liquid with 12ml complete medium at this time, while 3.75ul chloroquine (100mM, 4000 ×) is added.Change after liquid by Table 1 prepares calcium phosphate-DNA mixed liquor, stands 1 minute after reverse mixing for several times, is then rapidly added each ware cell with 3ml/ ware It in culture solution, shakes well while adding, rapidly joins dropwise.
Table 1 is calcium phosphate-DNA mixture system formula
12 hours after transfection, cell confluency degree should be close to 100%, and each ware changes liquid with 12ml fresh culture at this time, together When 90 μ l sodium butyrates (1M, 100 ×) are added.About 48 hours after transfection, whole 34ml HEK293T cell supernatant is collected, with It is packed into 50ml centrifuge tube after 0.45 μm of filter filtering, 2 ratio of according to the form below is added PEG-NaCl-PBS mixed liquor, is mixed by inversion It is placed afterwards in 4 DEG C 1.5 hours, mixes within during which every 20~30 minutes once or stood overnight after being mixed by inversion in 4 DEG C.
Table 2
By mixed liquor in 4 DEG C, 7000g is centrifuged 10 minutes, and visible white precipitates on tube wall, careful to remove whole supernatants, The fresh culture of appropriate volume (300ul~3ml) is added, jiggling makes precipitating dissolution to get QY-PhD-CAR molecule is arrived The viral concentration liquid of recombination saves after using or dispense immediately in -80 DEG C.
QY-PhD-CAR is in CD8+Expression in T cell: the separating peripheral blood mononuclear cells from peripheral blood sample, Then pass through the CD8 antibody separation and concentration CD8 of couple biotin+T lymphocyte counts, centrifugation.It is cultivated completely with RPMI1640 Base weight is outstanding, then presses 2 × 106The cell concentration of/ml, uniformly paving is into tissue culture plate.Utilize anti-CD3 (final concentration of 1 μ G/ml), anti-CD28 (final concentration of 1 μ g/ml) antibody and IL2 (final concentration of 10ng/ml) stimulate cell, effect After 48 hours, cell, the transduction for QY-PhD-CAR pseudovirus are collected.
The good target cell suspension of appropriate growth conditions is taken, 300g in centrifuge tube is put into and is centrifuged 5 minutes.It discards supernatant Liquid, with 1ml/1 × 106Pseudovirus concentrate is added in the ratio of cell, while the Polybrene of 8 μ g/ml of final concentration is added, gently Mixing is played in featheriness.Cell suspension is moved into culture dish, 37 DEG C, 350 × g centrifugation 90 minutes after centrifugation, are put back in incubator Continue to cultivate.After about 8~12 hours, the second wheel infection is carried out, step is same as above, and after about 12 hours, liquid is changed in centrifugation, is washed away with PBS Cell is resuspended with fresh culture in virus in culture medium, and the IL-2 and IL-7 that final concentration is 10ng/ml is added and keeps thin Born of the same parents' state.Continue to cultivate and pass in time, further amplifying cells.Metainfective 3rd day, according to cell state and proliferation feelings Condition adds complete RMPI1640 culture medium to cell, and cell concentration maintains 2 × 106/ml.And IL-2 and IL-7 are supplemented, it is dense eventually Degree is 10ng/ml.Metainfective 5th day, continue amplifying cells according to the 3rd day process.And utilize fluorescence quantitative PCR detection The expression of CD3 ζ determines the efficiency of infection of QY-PhD-001-CAR, QY-PhD-002-CAR and QY-PhD-003-CAR, usually needs Ensure that infection rate reaches 50% or more.Metainfective 5th day, cell detected for subsequent experimental or continues to cultivate and freeze.
For convenience's sake, it tests below and is called QY-PhD-CAR-T cell.
QY-PhD-CAR-T cell tests the specific killing of expression HIV-1 envelope protein cell
It is killed to further verify the specificity of QY-PhD-001-CAR, QY-PhD-002-CAR and QY-PhD-003-CAR Wound expression HIV-1 envelope protein cell effect.QY-PhD-CAR-T cell newly developed is compared simultaneously and inventor studies hair before The effect (Liuet al, Journal of Virology, 90:9712-9724.2016) (Fig. 3) of the VC-CAR-T cell of table, Inventor is then thin with the target of expression HIV-1 envelope protein by the QY-PhD-CAR-T cell or VC-CAR-T cell after transduction Born of the same parents are (Jurkat-gp160NL4-3) mixing, cell killing experiment is carried out in 96 orifice plates at U-shaped bottom.Target cell numbers are 104/ Hole, RMPI1640 complete medium volume are 150 holes μ l/.
In given effect target than (40:1 to 1.25:1) after 24 hours, passes through detection lactic dehydrogenase (LDH) in range Release, with each experimental group of determination by transformation CD8+The killing activity of CAR-T lymphocyte.
As the result is shown: in the effect target from 40:1 to 1.25:1 than in interval range, QY-PhD-001-CAR-T, QY-PhD- The target of 002-CAR-T and QY-PhD-003-CAR-T cell significantly killing expression HIV-1gp120 in a manner of dose-dependent is thin Born of the same parents (Jurkat-gp160NL4-3), and to control target cell (Jurkat-GFP) not significant fragmentation effect, and QY-PhD- The specific killing effect of CAR-T cell is suitable with VC-CAR-T cell.The result illustrates the QY-PhD-CAR-T that screening obtains Cell is the lethal effect mediated by the gp120 of identification target cell surface to target cell, and lethal effect is specific (see Fig. 4 and Fig. 5).
SEQUENCE LISTING
<110>the city biological medicine technology Co., Ltd of Shenzhen's regeneration
<120>inhibit the scFv sequence and its application of HIV-1 virus infection
<130>
<160> 10
<170> PatentIn version 3.5
<210> 1
<211> 342
<212> PRT
<213>HIV-1 neutralizing antibody
<400> 1
Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Thr Ala Ile Ile Ser Cys Arg Ser Leu Trp Tyr Gln Gln Arg Pro
20 25 30
Gly Gln Ala Pro Arg Leu Val Ile Tyr Leu Gln Tyr Pro Ala Gly Pro
35 40 45
Leu Leu Gly Ile Pro Asp Arg Phe Ser Gly Ser Arg Trp Gly Pro Asp
50 55 60
Tyr Asn Leu Thr Ile Ser Asn Leu Glu Ser Gly Asp Phe Gly Val Tyr
65 70 75 80
Tyr Cys Arg Leu Glu Gly Ser Pro Pro Val Asn Phe Gly Gln Gly Thr
85 90 95
Lys Val Gln Val Asp Ile Lys Arg Gln Val Gln Leu Val Gln Ser Gly
100 105 110
Gly Gln Met Lys Lys Pro Gly Glu Ser Met Arg Ile Ser Cys Arg Ala
115 120 125
Ser Arg Ser Pro Thr Arg Asn Ser His Arg Leu Ala Arg Leu Leu His
130 135 140
Leu Thr Pro Arg Leu His Gln Leu Thr Glu Pro Pro Pro Pro Glu Glu
145 150 155 160
Pro Pro Pro Pro Glu Glu Pro Pro Pro Pro Asp Arg Leu Met Ser Thr
165 170 175
Trp Thr Leu Val Pro Trp Pro Asn Thr Gly Thr Ala Arg Gln Thr Pro
180 185 190
Lys Ser Pro Asp Ser Arg Leu Leu Met Val Arg Leu Ser Gly Pro His
195 200 205
Arg Leu Pro Leu Asn Leu Ser Gly Met Pro Leu Ala Ile Asn Gly Gly
210 215 220
Ile Met Thr Ala Trp Gly Pro Gly Arg Ala Ser Val Asp Thr Ser Gly
225 230 235 240
Ser Pro Trp Gly Val Thr Arg Asp Lys Arg Trp Leu Phe Pro Leu Glu
245 250 255
Thr Lys Thr Gly Cys Leu Glu Thr Val Ser Thr Gln Phe Pro Trp Pro
260 265 270
Ala Gly Pro His Arg Lys Glu Gln Leu Lys Glu Leu Arg Ile Ile Ile
275 280 285
Phe Ser Arg Lys Ser Pro Lys Lys Arg Leu Gln Ser Leu Ala Ser Trp
290 295 300
Leu Phe Pro Val Asn Cys Tyr Pro Leu Thr Ile Pro His Asn Ile Arg
305 310 315 320
Ala Gly Ser Ile Lys Gly Lys Ala Trp Gly Ala Trp Gly Arg Gly Thr
325 330 335
Pro Val Ile Val Ser Ser
340
<210> 2
<211> 357
<212> PRT
<213>HIV-1 neutralizing antibody
<400> 2
Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Thr Ala Ile Ile Ser Cys Ser Pro Val Val Met Gln Trp Tyr Gln
20 25 30
Gln Arg Pro Gly Gln Ala Pro Arg Leu Val Ile Tyr Tyr Gly Phe Ile
35 40 45
Pro Ser Gly Leu Ala Ser Ile Gly Ile Pro Asp Arg Phe Ser Gly Ser
50 55 60
Arg Trp Gly Pro Asp Tyr Asn Leu Thr Ile Ser Asn Leu Glu Ser Gly
65 70 75 80
Asp Phe Gly Val Tyr Tyr Cys Gly Thr Pro Gly Arg Tyr Arg Glu Pro
85 90 95
Val Phe Gly Gln Gly Thr Lys Val Gln Val Asp Ile Lys Arg Gln Val
100 105 110
Gln Leu Val Gln Ser Gly Gly Gln Met Lys Lys Pro Gly Glu Ser Met
115 120 125
Arg Ile Ser Cys Arg Ala Ser Arg Ser Pro Thr Arg Asn Ser His Arg
130 135 140
Leu Ala Arg Leu Leu His Leu Thr Pro Arg Leu His Gln Leu His Leu
145 150 155 160
Arg Ala Ala Ala Ala Arg Arg Ala Ala Ala Ala Arg Arg Ala Ala Ala
165 170 175
Ala Arg Ser Leu Asn Val Asp Leu Asp Leu Gly Pro Leu Ala Lys Ser
180 185 190
Ser Ser Tyr Cys Ala Ile Ile Asn Thr Lys Ile Ser Arg Leu Gln Val
195 200 205
Ala Asp Gly Glu Ile Val Val Trp Pro His Arg Leu Pro Leu Asn Leu
210 215 220
Ser Gly Met Pro Met Cys Ile Gln Arg Arg Glu Met Thr Ser Leu Gly
225 230 235 240
Ala Trp Pro Gly Leu Cys Tyr His Ala Asp Leu Leu Lys Gly Asp Thr
245 250 255
Gln Glu Met Met Ala Val Ser Pro Gly Asp Lys Asp Arg Val Pro Gly
260 265 270
Asp Cys Val Lys Gln Phe Pro Trp Pro Ala Gly Pro His Arg Lys Glu
275 280 285
Gln Leu Lys Glu Leu Arg Ile Ile Ala Ser Val Asp Thr Thr Gln Thr
290 295 300
Cys Arg Gly Glu Thr His Lys Arg Trp Leu Phe Pro Leu Glu Thr Lys
305 310 315 320
Thr Gly Cys Leu Glu Thr Val Ser Asn Asn Phe Pro Trp Pro Ser Leu
325 330 335
Gly Arg Lys Arg Lys Glu Gln Leu Glu Glu Trp Gly Arg Gly Thr Pro
340 345 350
Val Ile Val Ser Ser
355
<210> 3
<211> 314
<212> PRT
<213>HIV-1 neutralizing antibody
<400> 3
Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Thr Ala Ile Ile Ser Cys Trp Leu Cys Trp Tyr Gln Gln Arg Pro
20 25 30
Gly Gln Ala Pro Arg Leu Val Ile Tyr Tyr His Leu Ala Leu Arg Pro
35 40 45
Phe Pro Arg Gly Gly Ile Pro Asp Arg Phe Ser Gly Ser Arg Trp Gly
50 55 60
Pro Asp Tyr Asn Leu Thr Ile Ser Asn Leu Glu Ser Gly Asp Phe Gly
65 70 75 80
Val Tyr Tyr Cys Gly Ile Val Ser Pro Leu Glu Ala Arg Gln Phe Gly
85 90 95
Gln Gly Thr Lys Val Gln Val Asp Ile Lys Arg Gln Val Gln Leu Val
100 105 110
Gln Ser Gly Gly Gln Met Lys Lys Pro Gly Glu Ser Met Arg Ile Ser
115 120 125
Cys Arg Ala Ser Glu Ile Leu Ile Asp Ser Pro Gly Phe Phe Ile Pro
130 135 140
Pro Asp Cys Thr Ser Cys Thr Glu Pro Pro Pro Pro Glu Glu Pro Pro
145 150 155 160
Pro Pro Glu Glu Pro Pro Pro Pro Asp Arg Leu Met Ser Thr Trp Thr
165 170 175
Leu Val Pro Trp Pro Asn Arg Arg Leu Ile Ala Gln Thr Pro Lys Ser
180 185 190
Pro Asp Ser Arg Leu Leu Met Val Arg Leu Ser Gly Pro Thr Asp Cys
195 200 205
Arg Gly Thr Cys Leu Gly Cys Arg Cys Val Ser Ser Asp Glu Asn Arg
210 215 220
Arg Ala Trp Gly Pro Gly Arg Ala Ser Val Asp Thr Thr Gln Thr Cys
225 230 235 240
Arg Gly Glu Thr His Lys Arg Trp Leu Phe Pro Leu Glu Thr Lys Thr
245 250 255
Gly Cys Leu Glu Thr Val Ser Asn Asn Phe His Gly Arg Leu Gly Arg
260 265 270
Ile Glu Arg Asn Asn Arg Asn Cys Glu Phe Phe His Val Glu Asn Leu
275 280 285
Gln Lys Glu Ala Pro Ser Leu Ala Ser Trp Ser Leu Val Pro Glu Trp
290 295 300
Gly Arg Gly Thr Pro Val Ile Val Ser Ser
305 310
<210> 4
<211> 350
<212> PRT
<213>HIV-1 neutralizing antibody
<400> 4
Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Thr Ala Ile Ile Ser Cys Trp Arg Ser Trp Tyr Gln Gln Arg Pro
20 25 30
Gly Gln Ala Pro Arg Leu Val Ile Tyr Leu Phe Gln Ile Lys Pro Pro
35 40 45
Gly Ile Lys Leu Gly Gly Ile Pro Asp Arg Phe Ser Gly Ser Arg Trp
50 55 60
Gly Pro Asp Tyr Asn Leu Thr Ile Ser Asn Leu Glu Ser Gly Asp Phe
65 70 75 80
Gly Val Tyr Tyr Cys Thr Ile Arg Leu Leu Lys Lys Pro Asp Lys Phe
85 90 95
Gly Gln Gly Thr Lys Val Gln Val Asp Ile Lys Arg Gln Val Gln Leu
100 105 110
Val Gln Ser Gly Gly Gln Met Lys Lys Pro Gly Glu Ser Met Arg Ile
115 120 125
Ser Cys Arg Ala Ser Lys Phe Ser Ser Thr Arg Gln Ala Ser Ser Ser
130 135 140
Asp Pro Gln Thr Ala Pro Ala Ala Pro Glu Ser Arg Arg Arg Gln Lys
145 150 155 160
Ser Arg Arg Arg Gln Ser Leu Asn Val Asp Leu Asp Leu Gly Pro Leu
165 170 175
Ala Lys Arg Gly Leu Asn Tyr Ala Ile Ile Asn Thr Lys Ile Ser Arg
180 185 190
Leu Gln Val Ala Asp Gly Glu Ile Val Val Trp Pro Pro Pro Thr Ala
195 200 205
Ala Glu Pro Val Trp Asp Ala Lys Lys Gly Leu Leu Met Lys Ile Asp
210 215 220
Asp Glu Pro Gly Gly Leu Ala Gly Pro Leu Leu Ile Pro Ala Pro Arg
225 230 235 240
Asn Val Leu Thr Gly Thr Arg Asp Asp Gly Cys Phe Pro Trp Arg Gln
245 250 255
Arg Gln Gly Ala Trp Arg Leu Cys Gln His Asn Ser Met Ala Gly Trp
260 265 270
Ala Ala Lys Gly Thr Thr Lys Gly Ile Ala Asn Asn Asn Phe Phe Thr
275 280 285
Leu Lys Ile Ser Lys Lys Lys Ala Pro Lys Leu Gly Val Ile Met Val
290 295 300
Ile Ala Val Ser Cys Val Lys Leu Leu Ser Ala His Asn Ser Thr Gln
305 310 315 320
His Thr Ser Arg Lys His Lys Val Ser Leu Gly Cys Leu Met Ser Glu
325 330 335
Leu Thr His Trp Gly Arg Gly Thr Pro Val Ile Val Ser Ser
340 345 350
<210> 5
<211> 357
<212> PRT
<213>HIV-1 neutralizing antibody
<400> 5
Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Thr Ala Ile Ile Ser Cys Phe Cys Val Cys Glu Trp Tyr Gln Gln
20 25 30
Arg Pro Gly Gln Ala Pro Arg Leu Val Ile Tyr His Gly His Ser Phe
35 40 45
Arg Ala Ser Arg Leu Val Gly Gly Ile Pro Asp Arg Phe Ser Gly Ser
50 55 60
Arg Trp Gly Pro Asp Tyr Asn Leu Thr Ile Ser Asn Leu Glu Ser Gly
65 70 75 80
Asp Phe Gly Val Tyr Tyr Cys Thr Asn Pro Ser Ile Lys Leu Glu Thr
85 90 95
Pro Phe Gly Gln Gly Thr Lys Val Gln Val Asp Ile Lys Arg Gln Val
100 105 110
Gln Leu Val Gln Ser Gly Gly Gln Met Lys Lys Pro Gly Glu Ser Met
115 120 125
Arg Ile Ser Cys Arg Ala Ser Pro Pro Lys Arg Ser Pro Thr Arg Asn
130 135 140
Ser His Arg Leu Ala Arg Leu Leu His Leu Thr Pro Arg Leu His Gln
145 150 155 160
Leu His Leu Arg Ala Ala Ala Ala Arg Arg Ala Ala Ala Ala Arg Arg
165 170 175
Ala Ala Ala Ala Arg Ser Leu Asn Val Asp Leu Asp Leu Gly Pro Leu
180 185 190
Ala Lys Pro Leu Gly Leu Asn Ala Ile Ile Asn Thr Lys Ile Ser Arg
195 200 205
Leu Gln Val Ala Asp Gly Glu Ile Val Val Trp Pro Pro Pro Thr Ala
210 215 220
Ala Glu Pro Val Trp Asp Ala Pro Trp Pro Gly Gly Glu Ala Ile Asp
225 230 235 240
Asp Glu Pro Gly Gly Leu Ala Gly Pro Leu Leu Ile Ser Leu Leu Cys
245 250 255
Val Arg Leu Lys Ala Thr Arg Asp Asp Gly Cys Phe Pro Trp Arg Gln
260 265 270
Arg Gln Gly Ala Trp Arg Leu Cys Gln His Asn Phe His Gly Arg Leu
275 280 285
Gly Arg Ile Glu Arg Asn Asn Arg Asn Cys Glu Phe Phe His Val Glu
290 295 300
Asn Leu Gln Lys Lys Gly Ser Lys Ala Trp Arg Asn His Gly His Ser
305 310 315 320
Cys Phe Leu Cys Glu Ile Val Ile Arg Ser Gln Phe His Thr Thr Tyr
325 330 335
Glu Pro Glu Ala Ser Val Lys Pro Gly Val Trp Gly Arg Gly Thr Pro
340 345 350
Val Ile Val Ser Ser
355
<210> 6
<211> 341
<212> PRT
<213>HIV-1 neutralizing antibody
<400> 6
Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Thr Ala Ile Ile Ser Cys Ser Gly His Arg Trp Tyr Gln Gln Arg
20 25 30
Pro Gly Gln Ala Pro Arg Leu Val Ile Tyr Tyr His Leu Ala Leu Arg
35 40 45
Pro Phe Pro Arg Gly Gly Ile Pro Asp Arg Phe Ser Gly Ser Arg Trp
50 55 60
Gly Pro Asp Tyr Asn Leu Thr Ile Ser Asn Leu Glu Ser Gly Asp Phe
65 70 75 80
Gly Val Tyr Tyr Cys Arg Ile Gln Ala Leu Gln Val Gly Ile Val Phe
85 90 95
Gly Gln Gly Thr Lys Val Gln Val Asp Ile Lys Arg Gln Val Gln Leu
100 105 110
Val Gln Ser Gly Gly Gln Met Lys Lys Pro Gly Glu Ser Met Arg Ile
115 120 125
Ser Cys Arg Ala Ser Ser Pro Leu Glu Ala Arg Gln Glu Ile Leu Ile
130 135 140
Asp Ser Pro Gly Phe Phe Ile Pro Pro Asp Cys Thr Ser Cys Thr Glu
145 150 155 160
Pro Pro Pro Pro Glu Glu Pro Pro Pro Pro Glu Glu Pro Pro Pro Pro
165 170 175
Asp Arg Leu Met Ser Thr Trp Thr Leu Val Pro Trp Pro Asn Arg Arg
180 185 190
Leu Ile Ala Gln Thr Pro Lys Ser Pro Asp Ser Arg Leu Leu Met Val
195 200 205
Arg Leu Ser Gly Pro Thr Asp Cys Arg Thr Cys Leu Gly Cys Arg Cys
210 215 220
Val Ser Ser Asp Glu Asn Arg Arg Ala Trp Gly Pro Gly Arg Ala Ser
225 230 235 240
Val Asp Thr Thr Gln Thr Cys Arg Gly Glu Thr His Lys Arg Trp Leu
245 250 255
Phe Pro Leu Glu Thr Lys Thr Gly Cys Leu Glu Thr Val Ser Asn Asn
260 265 270
Phe His Gly Arg Leu Gly Arg Ile Glu Arg Asn Asn Arg Asn Cys Glu
275 280 285
Phe Phe His Val Glu Asn Leu Gln Lys Arg Gly Ser Lys Ala Trp Arg
290 295 300
Asn His Gly His Ser Cys Phe Leu Cys Glu Ile Val Ile Arg Ser Gln
305 310 315 320
Phe His Thr Thr Tyr Glu Pro Glu Ala Ser Trp Gly Arg Gly Thr Pro
325 330 335
Val Ile Val Ser Ser
340
<210> 7
<211> 355
<212> PRT
<213>HIV-1 neutralizing antibody
<400> 7
Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Thr Ala Ile Ile Ser Cys Ser Gly His Arg Leu Phe Phe Leu Trp
20 25 30
Tyr Gln Gln Arg Pro Gly Gln Ala Pro Arg Leu Val Ile Tyr His Ser
35 40 45
Val Val Ser Leu Asp Ala Leu Leu Ile Gly Gly Ile Pro Asp Arg Phe
50 55 60
Ser Gly Ser Arg Trp Gly Pro Asp Tyr Asn Leu Thr Ile Ser Asn Leu
65 70 75 80
Glu Ser Gly Asp Phe Gly Val Tyr Tyr Cys Gly Gly Thr Arg Arg Val
85 90 95
Lys Arg Ser Pro Phe Gly Gln Gly Thr Lys Val Gln Val Asp Ile Lys
100 105 110
Arg Gln Val Gln Leu Val Gln Ser Gly Gly Gln Met Lys Lys Pro Gly
115 120 125
Glu Ser Met Arg Ile Ser Cys Arg Ala Ser Thr Arg Asn Ser His Arg
130 135 140
Leu Ala Arg Leu Leu His Leu Thr Pro Arg Leu His Gln Leu His Leu
145 150 155 160
Arg Ala Ala Ala Ala Arg Arg Ala Ala Ala Ala Arg Arg Ala Ala Ala
165 170 175
Ala Arg Ser Leu Asn Val Asp Leu Asp Leu Gly Pro Leu Ala Lys Phe
180 185 190
Pro Val Ser Ala Ile Ile Asn Thr Lys Ile Ser Arg Leu Gln Val Ala
195 200 205
Asp Gly Glu Ile Val Val Trp Pro Pro Pro Thr Ala Ala Glu Pro Val
210 215 220
Trp Asp Ala Tyr Asn Ala Lys Val Glu Gly Ile Asp Asp Glu Pro Gly
225 230 235 240
Gly Leu Ala Gly Pro Leu Leu Ile Pro Ser Arg Val Ser Ser Asn Ile
245 250 255
Ser Leu Thr Arg Asp Asp Gly Cys Phe Pro Trp Arg Gln Arg Gln Gly
260 265 270
Ala Trp Arg Leu Cys Gln His Asn Phe His Gly Arg Leu Gly Arg Ile
275 280 285
Glu Arg Asn Asn Arg Asn Cys Glu Phe Phe His Val Glu Asn Leu Gln
290 295 300
Lys Lys Gly Ser Lys Ala Trp Arg Asn His Gly His Ser Cys Phe Leu
305 310 315 320
Cys Glu Ile Val Ile Arg Ser Gln Phe His Thr Thr Tyr Glu Pro Glu
325 330 335
Ala Ser Val Lys Pro Gly Val Pro Trp Gly Arg Gly Thr Pro Val Ile
340 345 350
Val Ser Ser
355
<210> 8
<211> 355
<212> PRT
<213>HIV-1 neutralizing antibody
<400> 8
Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Thr Ala Ile Ile Ser Cys Glu Thr Gly Gly Trp Tyr Gln Gln Arg
20 25 30
Pro Gly Gln Ala Pro Arg Leu Val Ile Tyr Glu Ser Trp Ser Leu Phe
35 40 45
Gln Ile Lys Pro Pro Gly Gly Ile Pro Asp Arg Phe Ser Gly Ser Arg
50 55 60
Trp Gly Pro Asp Tyr Asn Leu Thr Ile Ser Asn Leu Glu Ser Gly Asp
65 70 75 80
Phe Gly Val Tyr Tyr Cys Thr Tyr Arg Val Thr Ile Arg Leu Leu Lys
85 90 95
Phe Gly Gln Gly Thr Lys Val Gln Val Asp Ile Lys Arg Gln Val Gln
100 105 110
Leu Val Gln Ser Gly Gly Gln Met Lys Lys Pro Gly Glu Ser Met Arg
115 120 125
Ile Ser Cys Arg Ala Ser Lys Pro Asp Lys Lys Phe Ser Ser Thr Arg
130 135 140
Gln Ala Ser Ser Ser Asp Pro Gln Thr Ala Pro Ala Ala Pro Glu Ser
145 150 155 160
Arg Arg Arg Gln Lys Ser Arg Arg Arg Gln Ser Leu Asn Val Asp Leu
165 170 175
Asp Leu Gly Pro Leu Ala Lys Arg Gly Leu Asn Tyr Ala Ile Ile Asn
180 185 190
Thr Lys Ile Ser Arg Leu Gln Val Ala Asp Gly Glu Ile Val Val Trp
195 200 205
Pro Pro Pro Thr Ala Ala Glu Pro Val Trp Asp Ala Lys Lys Gly Leu
210 215 220
Leu Met Lys Ile Asp Asp Glu Pro Gly Gly Leu Ala Gly Pro Leu Leu
225 230 235 240
Ile Pro Ala Pro Arg Asn Val Leu Thr Gly Thr Arg Asp Asp Gly Cys
245 250 255
Phe Pro Trp Arg Gln Arg Gln Gly Ala Trp Arg Leu Cys Gln His Asn
260 265 270
Ser Met Ala Gly Trp Ala Ala Lys Gly Thr Thr Lys Gly Ile Ala Asn
275 280 285
Asn Asn Phe Phe Thr Leu Lys Ile Ser Lys Lys Lys Ala Pro Lys Leu
290 295 300
Gly Val Ile Met Val Ile Ala Val Ser Cys Val Lys Leu Leu Ser Ala
305 310 315 320
His Asn Ser Thr Gln His Thr Ser Arg Lys His Lys Val Ser Leu Gly
325 330 335
Cys Leu Met Ser Glu Leu Thr His Trp Gly Arg Gly Thr Pro Val Ile
340 345 350
Val Ser Ser
355
<210> 9
<211> 343
<212> PRT
<213>HIV-1 neutralizing antibody
<400> 9
Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Thr Ala Ile Ile Ser Cys Asp Trp Trp Ser Trp Tyr Gln Gln Arg
20 25 30
Pro Gly Gln Ala Pro Arg Leu Val Ile Tyr Lys Ala Ala Arg Tyr Lys
35 40 45
Thr Arg Asn Pro Arg Gly Gly Ile Pro Asp Arg Phe Ser Gly Ser Arg
50 55 60
Trp Gly Pro Asp Tyr Asn Leu Thr Ile Ser Asn Leu Glu Ser Gly Asp
65 70 75 80
Phe Gly Val Tyr Tyr Cys Gln Gly Asp Asp Pro Ala Pro Gln Glu Ala
85 90 95
Phe Gly Gln Gly Thr Lys Val Gln Val Asp Ile Lys Arg Gln Val Gln
100 105 110
Leu Val Gln Ser Gly Gly Gln Met Lys Lys Pro Gly Glu Ser Met Arg
115 120 125
Ile Ser Cys Arg Ala Ser Arg Gln Glu Ile Leu Ile Asp Ser Pro Gly
130 135 140
Phe Phe Ile Pro Pro Asp Cys Thr Ser Cys Thr Glu Pro Pro Pro Pro
145 150 155 160
Glu Glu Pro Pro Pro Pro Val Ala Cys Arg Pro Gly Pro Trp Ser Pro
165 170 175
Gly Gln Thr Trp Ala Gln Leu Cys Asn Asn Lys His Gln Asn Leu Pro
180 185 190
Thr Pro Gly Cys Trp Asp Cys Ser Leu Ala Pro Thr Asp Cys Arg Thr
195 200 205
Cys Leu Gly Cys Gln Lys Arg Ile Thr Asn Glu Asp Arg Arg Ala Trp
210 215 220
Gly Pro Gly Arg Ala Ser Val Asp Thr Ser Ser Ser Glu Arg Thr Asp
225 230 235 240
Leu Gly Tyr Lys Arg Trp Leu Phe Pro Leu Glu Thr Lys Thr Gly Cys
245 250 255
Leu Glu Thr Val Ser Thr Gln Phe His Gly Arg Leu Gly Arg Ile Glu
260 265 270
Arg Asn Asn Arg Asn Cys Glu Phe Phe His Val Glu Asn Leu Gln Lys
275 280 285
Lys Gly Ser Lys Ala Trp Arg Asn His Gly His Ser Cys Phe Leu Cys
290 295 300
Glu Ile Val Ile Arg Ser Gln Phe His Thr Thr Tyr Glu Pro Glu Ala
305 310 315 320
Ser Val Lys Pro Gly Val Pro Asn Glu Ala Asn Ser Trp Gly Arg Gly
325 330 335
Thr Pro Val Ile Val Ser Ser
340
<210> 10
<211> 306
<212> PRT
<213>CAR molecule intracellular domain
<400> 10
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Phe
1 5 10 15
Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg
20 25 30
Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg
35 40 45
Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly
50 55 60
Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr
65 70 75 80
Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His
85 90 95
Arg Asn Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn
100 105 110
Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr
115 120 125
Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Phe Ser Val Val
130 135 140
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
145 150 155 160
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
165 170 175
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg
180 185 190
Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn
195 200 205
Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg
210 215 220
Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro
225 230 235 240
Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala
245 250 255
Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His
260 265 270
Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp
275 280 285
Ala Leu His Met Gln Ala Leu Pro Pro Arg Asp Tyr Lys Asp Asp Asp
290 295 300
Asp Lys
305

Claims (10)

1. inhibiting the scFv albumen of HIV-1 virus infection, it is characterised in that: the sequence of the scFv albumen such as SEQ ID NO.:1 Shown in~SEQ ID NO.:9.
2. a kind of CAR molecule for inhibiting HIV-1 virus infection, the CAR molecule includes extracellular antigen binding domain and structure intracellular Domain, it is characterised in that: the extracellular antigen binding domain be scFv albumen, the sequence of the scFv albumen such as SEQ ID NO.:1~ Shown in SEQ ID NO.:9.
3. CAR molecule according to claim 2, it is characterised in that: the sequence of the intracellular domain such as SEQ ID NO.: Shown in 10.
4. a kind of CAR-T cell for inhibiting HIV-1 virus infection, the CAR-T cell transduction have at least one CAR molecule, institute Stating CAR molecule includes extracellular antigen binding domain and intracellular domain, it is characterised in that: the extracellular antigen binding domain has SEQ Albumen shown in ID NO.:1~SEQ ID NO.:9 any sequence.
5. CAR-T cell according to claim 4, it is characterised in that: the extracellular antigen binding domain has SEQ ID Sequence shown in NO.:1, SEQ ID NO.2 and SEQ ID any one of NO.5.
6. CAR-T cell according to claim 4, it is characterised in that: the knot intracellular of the CAR molecule of the CAR-T cell The sequence in structure domain is as shown in SEQ ID NO.:10.
7. CAR-T cell according to any one of claim 4 to 6, it is characterised in that: T cell CD8+T cell.
Application of the 8.CAR-T cell in the preparation that HIV-1 infection cell is removed in preparation, it is characterised in that: the CAR-T cell As described in any one of claim 4~7.
Application of the 9.scFv albumen in the CAR-T cell that building inhibits HIV-1 virus infection, it is characterised in that: the scFv The sequence of albumen is as shown in one of SEQ ID NO.:1~SEQ ID NO.:9.
10.scFv albumen is preparing the application in HIV-1 virus targeting preparation, it is characterised in that: the sequence of the scFv albumen As shown in one of SEQ ID NO.:1~SEQ ID NO.:9.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113533723A (en) * 2021-07-28 2021-10-22 中国医科大学附属第一医院 Marker of cell infected by HIV and/or SIV and application thereof

Citations (3)

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Publication number Priority date Publication date Assignee Title
WO2012065055A2 (en) * 2010-11-12 2012-05-18 The Rockefeller University Fusion proteins for hiv therapy
CN104004092A (en) * 2014-06-05 2014-08-27 深圳市第三人民医院 Single-gene-coded bivalent or multivalent specific anti-HIV (human immunodeficiency virus) immunoadhesin
CN108779161A (en) * 2015-09-22 2018-11-09 宾夕法尼亚大学董事会 Redirect method of the T cell to treat HIV infection

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012065055A2 (en) * 2010-11-12 2012-05-18 The Rockefeller University Fusion proteins for hiv therapy
CN103282385A (en) * 2010-11-12 2013-09-04 美国洛克菲勒大学 Fusion proteins for HIV therapy
CN104004092A (en) * 2014-06-05 2014-08-27 深圳市第三人民医院 Single-gene-coded bivalent or multivalent specific anti-HIV (human immunodeficiency virus) immunoadhesin
CN108779161A (en) * 2015-09-22 2018-11-09 宾夕法尼亚大学董事会 Redirect method of the T cell to treat HIV infection

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113533723A (en) * 2021-07-28 2021-10-22 中国医科大学附属第一医院 Marker of cell infected by HIV and/or SIV and application thereof

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